Superfund site xenobiotics and other environmental toxicants are human health hazards whose toxicity is, in part, associated with altered patterns of gene expression. The goal of this project is to provide molecular mechanisms and models for exposure, focusing on the classic xenobiotic receptors (XenRs) PXR and CAR, and their induction of gene networks encoding the Phase I, II and III clearance pathways. Accordingly, to define the chemical space of XenRs in response to environmental toxins, in Aim II we will initiate a comparative chemical library screen using high throughput (HT) cell based luciferase reporter assays. Recently, we have determined that the nuclear receptor ERalpha is capable of responding to anticoagulants, antibacterial and anti-inflammatory drugs thus identifying it as a candidate xenobiotic sensor. Therefore as part of this Aim we will include ERalpha in the above XenRs screen. Some of our HT screens will include extracts gathered from Superfund sites by the Research Translation Core. Comparative gene expression studies will be conducted in Aim II to establish the overlap of ERalpha dependent gene regulation with known PXR and CAR target genes. The in vivo relevance will be established using a humanized hPXR/hCAR reporter mouse. In Aim III we will determine how XenRs control the xenobiotic response at the genome-wide level. Chromatin immunoprecipitation coupled with massively parallel deep sequencing (ChlPSeq) will be used to identify PXR, CAR and ERalpha cistromes, before and after treatment with high affinity agonists to reveal unique and common (core) xenobiotic networks. The aggregate binding sites will comprise a xenobiotic cistrome. Finally, in Aim I, we describe a new HT screening platform called NHR Transcriptional Promoter Ontology which allows us to explore xenobiotic regulation by all human NHRs (+/- ligands) by screening against a panel of ~300 drug metabolism reporter constructs comprised of P450 and conjugation enzyme and transporter sets. This is a unique opportunity to redefine the molecular basis of NHR-xenobiotic regulation and will provide a new roadmap for future study. We will collaborate with the Research Translation Core and Community Engagement Core to share this work with our SRP tribal science partners, industry, EPA, and ATSDR.